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Intraoperative Cytology of Central Nervous System Lesions: A Diagnostic Approach Based on Pattern Recognition
Cytologic methods for the diagnosis of central nervous system (CNS) lesions have been used for about 50 years. Despite this, intraoperative consultation in most centers employs only traditional frozen section analysis as the sole means to render an intraoperative diagnosis. Although frozen section analysis, in general, has the advantage of architectural preservation, the superimposed artifacts of freezing tissue occasionally limit and distort important diagnostic features of some lesions. This is particularly true when freezing CNS lesions as many lesions typically have interstitial edema, contributing to “slow freeze” artifact. Nuclear details including the size and shape of nuclei are often distorted by freezing the tissue. Intraoperative cytology evaluation yields superior cytologic detail of cells and nuclei and is devoid of the artifacts associated with frozen section analysis. When cytology and frozen section are used in tandem, the cytologic details of the lesion and architectural features are preserved, which in turn yields superior intraoperative interpretation and accuracy, assisting the neurosurgeon to proceed with appropriate treatment for the patient.
Definition of “Practical Pattern Recognition−Based Approach” to Intraoperative Central Nervous System Cytology
A useful approach for intraoperative neuropathology cytology diagnosis is to first categorize lesions that exhibit one or more cytologic low-power smear patterns at low to intermediate magnification (×4, ×10, ×20) and then confirm individual cytologic features at high magnification (×40 to ×60), correlating the low-power pattern recognition impressions with the high-power cytologic features to reach a final diagnosis or differential diagnosis. We characterize this as a “practical algorithmic pattern recognition” approach to CNS cytologic smear diagnosis as outlined in Chapter 1 of this book. The set of smear patterns that we discuss in the following sections are reliant on specific cell types and the structural relations of the cells to each other, and they provide clues to the intraoperative diagnosis of the pathologic disease. For example, high-grade astrocytoma and medulloblastoma may appear similar on frozen section, but by cytologic smear analysis, the pattern of spread, the cellular relationship to stromal elements, and individual cytologic details are sufficiently distinct to differentiate them from one another.
These patterns do not necessarily rely on the presence of a specific cell type, for instance, astrocytes in a case of an astrocytoma. A cytologic pattern may include a specific type of architectural structure that the tumor cells and associated stromal cells or blood vessels exhibit, such as a papillary structure. Another pattern may encompass the interrelationship that clusters of neoplastic cells have with surrounding blood vessels, stromal tissue, extracellular matrix, or inflammatory cells.
The pattern recognition approach requires each intraoperative consultation to be attacked in the same way so that establishing a routine keeps one safe in the treacherous minefield of neuropathology intraoperative consultation. This cannot be emphasized enough. After preparing an evenly spread smear slide, the cytopathologist should always start at a low-power magnification to assess the pattern made up of the various elements and their relationship to one another and then proceed to higher magnification to confirm the cell types present. This approach will provide a consistent, accurate, and valuable intraoperative neuropathology interpretation.
As described in Chapter 1 , this method emphasizes grouping together pathologic processes with an element or elements that they have in common and then subcategorizing the lesions within a pattern category by their more specific cellular characteristics. Although this approach may be intuitive to many experienced cytopathologists, in the heat of the moment, particularly in neuropathology, it is all too easy to forget these guidelines and rush to high-power magnification, immediately incurring the risk of “missing the forest for the trees.”
Method of Central Nervous System Squash Preparation
The concept of squash preparation is simple but difficult to master unless one uses the following technique consistently as detailed. We prefer to use the one-step method to prepare a smear slide by taking a small piece of tissue with a scalpel blade (about the size of a pinhead), placing the material toward the label end of the slide, and then smearing the specimen with another slide, holding the second slide at a 45-degree angle to the first and applying even pressure during smearing. If a stereotactic core biopsy is submitted, it is best to section a pinhead-sized portion from both poles of the core biopsy specimen and to place both tissue fragments on the same side, yielding two cellular “flares” on that slide. The two-flare technique allows the microscopist to virtually compare both ends of the core biopsy rapidly. If both cytologic flares are nondiagnostic, the chance of diagnostic material being present within the nonsampled portion of the core biopsy is low. Consequently, the surgeon should be advised to submit more tissue for evaluation when this scenario arises. If only one flare area has lesional tissue, there is a good possibility that the surgeon has sampled the edge of the lesion. In this situation, a rebiopsy may be indicated to obtain adequate material for intraoperative and permanent assessment.
We have found that frozen section is not always necessary and, at times, is contraindicated, especially when only a limited amount of biopsy tissue is available at time of intraoperative consultation. If a preliminary diagnosis is unattainable by cytology alone, a frozen section of a half of the core biopsy may be helpful in reaching a preliminary diagnosis. Refrain from freezing the entire biopsy specimen. Punctate areas of hemorrhage are often the most useful areas for intraoperative evaluation.
Cytologic smears of mesenchymal types of specimen (e.g., meningiomas) are best prepared by scraping the scalpel blade across the cut surface and placing the scraped liquefied tissue on the glass slide to be smeared.
Once a tissue sample is smeared, the slide should be immediately fixed in alcohol and rapidly stained with H&E. Rarely, the same slide preparation technique can be used to prepare an air-dried slide to be stained with a Giemsa stain, which can highlight lymphoid cells and macrophages.
The most common artifacts include crush, excessive clumping, and air-drying artifact, all leading to diminished cytologic detail. It is important not to smear too much of the specimen on one slide, which may yield a slide too thick for optimal cytologic detail. Essential details of neuropathology cytologic diagnosis are dramatically dependent on the correct procedure to create a slide with a monolayer of tissue. Recognition of the fine fibrillary processes that are often associated with glial tumors is dependent on a thinly smeared specimen.
Advantages and Limitations of Cytologic Smear Preparation
Cytologic Smears: Advantages
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Speed
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Ease and reduplication of preparation
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Preservation of cytologic detail
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Optimal in small tissue samples
Cytologic Smears: Limitations
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Relies on tissue soft enough to smear
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Histologic architecture is limited
Normal Central Nervous System Constituents and Associated Pathologic Reactions
In order to render the finest intraoperative diagnosis, it is imperative to recognize normal CNS constituents that can be divided broadly into neuroectodermal and mesenchymal derivatives. Neurons, glia (astrocytes, oligodendrocytes, ependyma), and choroid plexus are neuroectodermally derived elements, while vasculature, meninges, and microglia (bone marrow–derived monocytes) are of mesenchymal derivation.
Neurons
Neurons are large, polygonal cells with low nuclear-to-cytoplasmic (N:C) ratios, a prominent centrally placed nucleus, and conspicuous rough endoplasmic reticulum (Nissl substance). They reside in the gray matter of the cortices and deep cortical nuclei, but a few are present in the white matter of the temporal lobe ( Fig. 11-1 ). Another type of neuron is the granular neuron. These are smaller, naked nuclei by light microscopy and are seen in the cerebellum and hippocampal formation ( Fig. 11-2 ). Immunoperoxidase studies (immunohistochemistry [IHC]) for synaptophysin (cytoplasmic/membranous) and NeuN (nuclear) will identify neurons.
Pathologic Reactions
In acute ischemic events, such as infarction, the neuron undergoes specific identifiable changes, the so-called “red neuron.” These conspicuous neurons have bright, eosinophilic cytoplasm with concomitant loss of the Nissl substance and dark, pyknotic nuclei, in which prominent nucleoli are not discernable. The red neuron is the sine qua non of ischemic or hypoxic change. The background will likely be infiltrated by numerous macrophages as well. An important guideline in intraoperative neuropathology diagnosis is “be very cautious in diagnosing a neoplasm in a background of numerous macrophages” because this may relate to an ischemic or inflammatory process.
Astrocytes
There are two types of astrocytes or “star cells”: the more common fibrillary astrocyte and the protoplasmic astrocyte. The fibrillary type is present in the white matter and shows numerous and prominent cytoplasmic extensions compared with the protoplasmic astrocytes that display round nuclei and are often located in the gray matter ( Fig. 11-3 ). Most astrocytic neoplasms arise from the fibrillary astrocyte and thus give rise to a fibrillary background in squash preparations. The protoplasmic astrocytes are present predominantly in the gray matter. IHC for glial fibrillary acidic protein (GFAP) and S100 are useful to distinguish glial lineage but are not specific for astrocytes per se.
Pathologic Reactions
Gliosis is a response to CNS injury and consists of two components, astrocytic hypertrophy and hyperplasia. The initial response, astrocytic hypertrophy, consists of an increase in cell size and cytoplasmic prominence. Astrocytes with well-defined astrocytic cytoplasm with multiple (more than two) cytoplasmic extensions usually indicate reactive gliosis.
Chronic astrocytosis is characterized by dense fibrillary gliosis. An increase in astrocytic numbers or hyperplasia may also follow insult to the CNS. Rare non-neoplastic mitoses may be observed, but a hyperplastic response is typified by the presence of “mirror” nuclei. These are astrocytic nuclei that occur in physically contiguous matched pairs.
Astrocytes with abundant eosinophilic cytoplasm and well-defined, rounded edges with few cytoplasmic extensions are referred to as gemistocytes or “stuffed” astrocytes; however, when abundant, these typify gemistocytic astrocytoma rather than a reactive gliotic process.
Creutzfeldt astrocytes are not specific for but often seen in demyelinating disease. These are reactive astrocytes with multiple, small nuclei (micronuclei). The precursors of these nuclei have tiny chromatid bodies termed granular mitoses . Although they are usually associated with reactive conditions, they are rarely associated with high-grade gliomas.
Rosenthal Fibers
Rosenthal fibers are brightly eosinophilic, ropy, elongate cytoplasmic inclusions seen in astrocytes cell processes and are possibly accumulations of several types of cytoplasmic proteins. Rosenthal fibers are seen in association with neoplastic, reactive (indicative of chronicity), or metabolic processes (Alexander disease). In neoplasia, Rosenthal fibers are often seen in pilocytic astrocytoma (PA). However, the presence of Rosenthal fibers does not exclusively imply neoplasia, as they may also be present in marked chronic gliosis, for example, in chronic gliotic tissue around a pineal gland cyst or craniopharyngioma.
Eosinophilic Granular Bodies
Eosinophilic granular bodies (EGB) are rounded, “granular,” bright eosinophilic bodies that share similar protein structure with Rosenthal fibers and are immunoreactive with GFAP, ubiquitin, and alpha-beta-crystallin. It is important to recognize EGB because they are particularly associated with three types of tumors: juvenile PA, pleomorphic xanthoastrocytoma (PXA), and ganglioglioma.
Corpora Amylacea
Corpora amylacea are intracytoplasmic glucose polymers (polyglucosan bodies) present in astrocytic cytoplasm. They are located around blood vessels and in subpial locations and may be a result of trauma, epilepsy, or normal aging.
Corpora stain positively for PAS, GMS, and alcian blue, and therefore care should be taken not to misinterpret these structures as fungal yeasts. The size of corpora amylacea and their variability in size are clues that aid in their distinction from yeasts.
Oligodendrocytes
Oligodendrocytes are myelin-producing glial cells associated with white matter. They are also seen in the gray matter, where they serve as “satellite cells” to neurons. Oligodendrocyte satellitosis may be physiologic or associated with neoplasia, most often with oligodendroglioma, and is known as a secondary structure of Scherer. Oligodendrocytes have few, if any, cytoplasmic processes compared with astrocytes and are typically seen as naked nuclei in the neuropil. Unlike astrocytomas, oligodendrogliomas invade neuropil without prominent disruption of myelin. Thus the background squash of an oligodendroglioma may be less fibrillary and more granular with some areas of ropy myelin. The nuclei of oligodendrocytes are small, uniformly round, and hyperchromatic. Occasionally they display a conspicuous nucleolus. The nuclei of astrocytes are more oval or oblong, mildly irregular, and more hyperchromatic.
A characteristic morphologic feature of oligodendroglia as a result of delayed fixation is referred to as perinuclear halos or a “fried egg” appearance and is seen in normal and neoplastic oligodendrocytes.
Pathologic Reactions
Oligodendrocytes demonstrate limited pathologic reactions, which are mainly proliferation with satellitosis.
Ependyma
Ependymal cells line the surface of the ventricular system and vary from the robust ciliated cells of the fetal system to the flattened cuboidal cells of adults. Subependymal glia is present just beneath the ependymal layer and gives rise to the subependymomas. Normal ependymal cells are GFAP negative and vimentin positive. Epithelial membrane antigen (EMA) is useful to detect ependymal differentiation because ependymomas may demonstrate perinuclear dot or ringlike immunoreactivity for EMA.
Tanycytes are applicable to several cell populations of the developing and adult nervous system and share a highly elongated shape that spans the entire surface of the ependyma from the ventricular system to the subependymal-CNS parenchyma. Tanycytes in adults are located in the floor of the third ventricle. Tanycytes are strongly GFAP positive and are thought to be precursors of the tanycytic ependymoma and astroblastoma.
Choroid Plexus
Choroid plexus comprises papillary tufts of epithelium-covering fibrovascular connective tissue that project into the ventricles. These epithelial cells are larger than ependymal cells and have a cobblestone surface. The largest masses of choroid plexus cells are found in the lateral ventricular atria and are called the glomerula choroidea. Choroid plexus cells are typically negative for GFAP and positive for S100 and transthyretin. Meningothelial rests and whorls are common inhabitants of normal choroid plexus, and their location explains the occurrence of intraventricular meningiomas.
Microglia/Monocytes/Macrophages
Microglia are bone marrow–derived monocytes that arrive via the bloodstream and transform into indigenous microglia of the CNS. Resting microglia blend inconspicuously into the neuropil and consist of naked nuclei on hematoxylin-eosin. Microglia show immunoreactivity for CD68 and CD163.
Pathologic Reactions
These include diffuse proliferation and infiltration of CNS parenchyma with microglial cells that can result in the formation of microglial nodules that consist of microglia and astrocytes. These are seen in viral and rickettsial infections. Proliferative microglia are seen as rod-shaped, haphazardly arranged nuclei.
Tissue macrophages may be present in many pathologic conditions including ischemia, demyelinating diseases, progressive multifocal leukoencephalopathy (PML), tuberculosis, histoplasmosis, extramedullary sinus histiocytosis, xanthomatous lesions, and primary CNS lymphomas treated with steroids, as well as in metastatic tumors with necrosis. They are uncommonly present in CNS gliomas, except when associated with areas of necrosis in a high-grade lesion. The presence of macrophages at the time of intraoperative consultation should give one pause before making a preliminary diagnosis of a primary glial neoplasm ( Fig. 11-4 ).
Importance of Clinical History and Neuroimaging Findings
To ensure that the intraoperative neuropathology experience is the least stressful for the pathologist and most beneficial to the neurosurgeon and patient, it is imperative that the clinical history and neuroimaging features of a case are reviewed before the tissue arrives in the surgical cutting room. The important information that should be obtained before proceeding with the intraoperative cytologic evaluation includes the age of the patient, location of the lesion, presenting symptoms, and neuroimaging features, including whether the lesion is well defined versus infiltrative and enhancing or nonenhancing.
Other useful information includes the type and duration of clinical symptoms. For example, a history of seizures is more in keeping with a slower-growing lesion such as a low-grade astrocytoma, as compared with a rapidly growing lesion such as a glioblastoma. Thorough discussion of the neuroimaging findings of CNS lesions can be found in these references.
Patterns in Central Nervous System Smear Cytology: Basic Principles
When evaluating a lesion by cytologic smear at low power, particular attention should be applied to the following:
- 1.
Relationship of neoplastic cells to blood vessels
- 2.
Blood vessel morphology: nonproliferative versus proliferative
- 3.
Background features: feltlike versus fibrillary—presence or absence of necrosis?
1
Relationship of Neoplastic Cells to Blood Vessels ( Plates 11-1A1-3; 11-1B1-3; 11-1C1-3 )
Gliomas, especially astrocytomas, usually demonstrate an aggregation of tumor cells close to blood vessels, the concentration of which decreases the farther away the tumor cells are from a blood vessel yielding a Perivascular Gradient Pattern (Pattern 1A). CNS lymphomas infiltrate and/or are intimately associated with blood vessel walls, and they are often dispersed in a discohesive randomized fashion farther away from blood vessels, yielding an Angiocentric and Diffuse Pattern (Pattern 1B) . Reactive lymphoid infiltrates may exhibit a similar pattern. Metastatic carcinomas often are characterized by tissue fragments of malignant cells distributed in a randomized fashion with or without an affinity to blood vessels yielding a Randomized Tissue Fragment With or Without Vascular Affinity Pattern (Pattern 1C).
Plate 11-1A
Pattern 1A Perivascular Gradient
Plate 11-1A Table
Pattern 1: Relationship of Neoplastic Cells With Blood Vessels
Entities in the Differential Diagnosis
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Plate 11-1B
Pattern 1B Angiocentric and Diffuse
Plate 11-1B Table
Pattern 1: Relationship of Neoplastic Cells With Blood Vessels
Entities in the Differential Diagnosis
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Plate 11-1C
Pattern 1C Randomized Tissue Fragments With or Without Vascular Affinity
Plate 11-1C Table
Pattern 1: Relationship of Neoplastic Cells With Blood Vessels
Entities in the Differential Diagnosis
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2
Blood Vessel Morphology: Nonproliferative Versus Proliferative ( Plates 11-2A1-3; 11-2B1-3 )
Grade 2 and 3 astrocytomas, pilomyxoid astrocytoma (PMA), oligodendrogliomas, ependymomas, reactive gliosis, and most metastatic carcinomas and lymphomas are associated with Thin-Walled Blood Vessels Without Endothelial Cell Proliferation (Nonproliferative) (Pattern 2A). In contrast, glioblastomas, some pilocytic astrocytomas, anaplastic oligodendrogliomas, and some metastatic carcinomas and lymphomas are associated with Blood Vessels with Endothelial Cell Proliferation (Proliferative) (Pattern 2B).
Plate 11-2A
Pattern 2A Thin-Walled Blood Vessels Without Endothelial Cell Proliferation (Nonproliferative)
Plate 11-2A Table
Pattern 2: Blood Vessel Morphology: Nonproliferative Versus Proliferative
Entities in the Differential Diagnosis
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Plate 11-2B
Pattern 2B Blood Vessels With Endothelial Cell Proliferation (Proliferative)
Plate 11-2B Table
Pattern 2: Blood Vessel Morphology: Nonproliferative Versus Proliferative
Entities in the Differential Diagnosis
|
3
Background Features: “Billiard Table/Feltlike” or Granular Versus Fibrillary ( Plates 11-3A1-3; 11-3B1-3 )
Normal brain matter, some oligodendrogliomas, metastatic tumors, and lymphomas usually show a “ Billiard Table/Feltlike” or Granular Background (Pattern 3A) on smear slides.
Plate 11-3A
Pattern 3A Billiard Table/Feltlike or Granular Background
Plate 11-3A Table
Pattern 3: Background Features: Billiard Table/Feltlike or Granular Versus Fibrillary/Threadlike
Entities in the Differential Diagnosis
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Plate 11-3B
Pattern 3B Fibrillary or Threadlike Background
Plate 11-3B Table
Pattern 3: Background Features: Billiard Table/Feltlike or Granular Versus Fibrillary/Threadlike
Entities in the Differential Diagnosis
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Reactive gliosis, astrocytomas, glioblastoma, and ependymomas often show a Fibrillary or Threadlike Background (Pattern 3B) on the smear slides. Oligodendrogliomas are often associated with an attenuated fibrillary background. Metastatic tumors and lymphomas associated with reactive gliosis may also be associated with a fibrillary background. Fibrillary smear backgrounds are best appreciated by removing the condenser and/or closing the annulus of the microscope.
Attention to the presence or absence of necrosis is also an important feature to observe while scanning the smear slide. Necrosis can be associated with inflammatory or infectious processes in addition to glioblastomas, anaplastic oligodendroglioma, medulloblastoma, metastatic tumors, and lymphoma.
The types of background cytologic features are useful for broadly categorizing lesions (e.g., astrocytoma vs. metastatic lesions).
Each lesion can have a type of pattern or subcategory of pattern in each of Patterns 1, 2, and 3; for example, a glioblastoma has a “perivascular gradient” Pattern 1A, in conjunction with a “thin blood vessel with endothelial proliferation (proliferative)” Pattern 2B and a “fibrillary background” Pattern 3B, creating a diagnostic overall pattern.
Additional Smear Patterns
There are additional cytologic smear patterns associated with some specific CNS and peripheral nervous system lesions, but these can also apply to a subset of metastatic lesions as well.
4
Vascular Coronal Pattern ( Plates 11-4A-C )
This pattern is characterized by a population of cells (ependymal, primitive neuroectodermal, epithelial, or melanocytic) that surround a blood vessel in a palisaded or corona-like fashion. This pattern is characteristic of ependymomas and choroid plexus papillomas but can also be seen with metastatic melanomas and variants of metastatic adenocarcinomas.
Plate 11-4
Pattern 4 Vascular Coronal
Plate 11-4 Table
Pattern 4: Vascular Coronal
Entities in the Differential Diagnosis
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5
Randomized Loose Tissue Fragments and Dispersed Single Cell Pattern ( Plates 11-5A-C )
This pattern is characterized by the presence or absence of loosely aggregated tissue fragments associated with a population of discohesive cells. Pituitary adenomas are often characterized by this pattern. Normal pituitary gland may show this pattern if smeared too aggressively. A touch preparation technique may be more appropriate when there is a suspected pituitary adenoma.
Plate 11-5
Pattern 5 Randomized Loose Tissue Fragment and Dispersed Single Cell
Other lesions of neural crest origin, such as metastatic melanoma and metastatic neuroendocrine carcinomas, can also show this pattern. Some metastatic adenocarcinomas such as metastatic lobular or “signet ring” cell carcinoma may exhibit this pattern.
6
Randomized Loose Tissue Fragment and Dispersed Small Cell Pattern ( Plates 11-6A-C )
This pattern is essentially identical to the aforementioned randomized loose tissue fragment and dispersed single cell pattern, except these entities are composed exclusively of small cells showing high nuclear to cytoplasmic ratios (N:C). This is a common presentation of primitive neuroectodermal tumors and medulloblastomas and metastatic small cell carcinomas.
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